ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of antigens or antibodies in a sample. It works by detecting the binding of an antigen or antibody to its complementary binding partner which is attached to an enzyme. This binding is detected through the enzyme's action on a substrate, producing a color change that can be measured. There are different types of ELISA including direct sandwich, indirect, and competitive ELISA. ELISA is widely used for detecting proteins, hormones, and other biomolecules and in applications like pregnancy and HIV testing.

2. And if you would count the graces of Allâh,
never could you be able to count them Truly!
Allâh is Oft-Forgiving, Most Merciful.
(Surah An-Nahl:18)
)

3. ELISA
ENZYME LINKED IMMUNOSORBENT ASSAY
By: Dr.MUHAMAD SALMAN PASHA

4. Enzyme-linked
immunosorbant assay
(ELISA) is a non-isotopic
immunoassay. An enzyme
is used as a label in ELISA

5. ELISA
Link:
https://www.youtube.com/watch?v=oyiz2lci4dY

6. Definitions
Antibodies (also known as
immunoglobulins abbreviated Ig) are
gamma globulin proteins that are found
in blood and are used by the immune
system to identify and neutralize foreign
objects, such as bacteria and viruses.

7. Antigens
A substance that when introduced into the
body
stimulates the production of an antibody
Immunoassay
A laboratory technique that makes use of
the binding between an antigen and its
homologous antibody in order to identify
and quantify the specific antigen or
antibody in a sample

9. Analyte
The sample being analyzed and
in immunoasssays the analyte
is either Antibody or Antigen

10. BASIC PRINCIPLES OF ELISA

11. ELISA is based on the immunochemical principles
of antigen-antibody reaction. The stages of ELISA
are summarized:
1. The antibody against the protein to be determined is
fixed on an inert solid such as polystyrene.
2. The biological sample containing the protein to be
estimated is applied on the antibody coated surface.
3. The protein antibody complex is then reacted with a
second protein specific antibody to which an enzyme is
covalently linked. These enzymes must be easily assayable
and produce preferably coloured products. Peroxidase,
amylase and alkaline phosphatase are commonly used.

12. 4. After washing the unbound antibody linked enzyme, the
enzyme bound to the second antibody complex is assayed.
5. The enzyme activity is determined by its action on a
substrate to form a product (usually coloured). This is
related to the concentration of the protein being
estimated. The principle for the use of the enzyme
peroxidase in ELISA is illustrated next.

14. Enzyme Product Product No.
(Total Assays ¹)
Absorban
ce
and Color
Detection
Limit ²
Primary
(1°) and
Secondary
(2°)
Antibody
Dilutions ²
AP PNPP
Substrat
e
1-
Step Sol
ution
Tablet
Kit
Tablets
(105)
Powder
(25g)
37621
(1000 wells)
37620
(5250 wells)
34047
(5250 wells)
34045
(250,000 wells)
405nm
Yellow
~10ng/well
(100ng/mL)
1° 1:500
2° 1:5K
to 1:20K

15. HRP ABTS Substrate
1-Step Solution
Tablets (50)
37615
(2000 wells)
34026
(3330 wells)
410nm
(650nm)
Green
~250pg/well
(2.5ng/mLl)
1° 1:1K
2° 1:5K
to 1:50K
Gal ONPG
Substrate
Powder (5g)
34055 410nm
Yellow
~10ng/well
(100ng/mL)
1° 1:500
2° 1:5K

16. Introduction
 The Antibody: An immunoglobulin, a
specialized immune protein, produced
because of the introduction of an
antigen into the body, and which
possesses the remarkable ability to
combine with the very antigen that
triggered its production (specific
affinity)
 The antibody recognises and bind to
the antigenic determinant region of
the antigen

17. INTRODUCTION

18. ELISA technique
Is a biochemical technique used mainly in
immunology to detect the presence of an
antibody or an antigen in a sample.
 The technique is divided into
1- Competitive ELISA
2- Sandwich ELISA (also called direct ELISA)
3- Indirect ELISA

19. Competitive ELISA
The labelled antigen
competes for primary antibody
binding sites with the sample
antigen (unlabeled). The more
antigen in the sample, the less
labelled antigen is retained in
the well and the weaker the
signal).

21. Sandwich ELISA
The ELISA plate is coated with
Antibody to detect specific
antigen

22. Sandwich ELISA

23. Indirect ELISA
 The protein antigen to be tested
for is added to each well of ELISA
plate, where it is given time to
adhere to the plastic through
charge interactions
 A solution of non-reacting protein
is added to block any plastic
surface in the well that remains
uncoated by the protein antigen

24. Indirect ELISA

25. DIRECT SANDWICH ELISA
An antigen coated to a multiwell plate is
detected by an antibody that has been
directly conjugated to an enzyme. This can
also be reversed, with an antibody coated to
the plate and a labeled antigen used for
detection, but the second option is less
common.
This type of elisa has two main advantages:
It is faster, since fewer steps are required.
It is less prone to error, since there are fewer
steps and reagents.

26. DIRECT SANDWICH ELISA

27. ELISA ANIMATION
https://www.youtube.com/watch?v=RRbuz3VQ100

28. Applications:
o ELISA is widely used for the determination of small
quantities of proteins (hormones, antigens, antibodies)
and other biological substances.
o The most commonly used pregnancy test for the
detection of human chorionic gonadotropin (hCG) in
urine is based on ELISA. By this test, pregnancy can be
detected within few days after conception.
o ELISA is also been used for the diagnosis of AIDS.

29. An example of an ELISA
experiment
Before starting the work read
kit instruction carefully
1- The 96 well plate is labeled
carefully and the first wells are
used to draw the standard
curve

30. The sample is added to plate
in duplicate or triplicate and
then the mean result is
calculated
The quality control sample
which is provided with the kit is
treated as the test samples

31. Results
After reading the results the
standard curve is drawn were
the concentration is blotted on
the X-axis and the
absorbance on the Y-axis
Concentration ng/ml
Absorption
nm

32. ELISA TEST